ALA-PDT activates macrophage autophagy via the ROS-EP300 pathway to kill intracellular Mycobacteroides abscessus

BACKGROUND: Mycobacteroides abscessus is a common rapidly growing non-tuberculosis mycobacteria (NTM) that exhibits resistance to most antibiotics and is associated with low cure rates, highlighting an urgent need for new therapeutic strategies. Our previous clinical study has found that ALA-PDT may represent a novel and promising approach for treating M.abscessus infection, although its precise mechanism of action remains to be elucidated. METHODS: To investigate the mechanism by which ALA-PDT kills intracellular M. abscessus, we established an intracellular infection model using THP-1 to evaluate its bactericidal effect. Subsequently, RNA-sequencing analysis and targeted in vitro experiments were performed to explore the underlying mechanisms. RESULTS: ALA-PDT significantly reduced the intracellular survival of M. abscessus in THP-1. RNA-sequencing revealed that ALA-PDT modulates multiple cellular pathways, notably inducing the upregulation of autophagy-related genes. Consistently, ALA-PDT increased autophagosome formation and LC3 expression in both infected and uninfected macrophages. The bactericidal effect of ALA-PDT against intracellular M.abscessus was markedly attenuated by an autophagy inhibitor, confirming the functional role of autophagy. In addition, ALA-PDT promoted the generation of reactive oxygen species (ROS), while a ROS inhibitor suppressed the ALA-PDT induced increase in LC3 expression and the decrease in intracellular bacterial survival. Transcriptomic analysis suggested that EP300 may play a key regulatory role in this process. In vitro experiments confirmed that ALA-PDT downregulated EP300 expression, and an EP300 activator significantly reversed the ALA-PDT-mediated increase in LC3 expression and reduction in intracellular bacteria. Finally, it was found that ALA-PDT can alter the overall acetylation levels in macrophages, pointing to a potential epigenetic mechanism. CONCLUSIONS: These findings demonstrate that ALA-PDT activates antibacterial autophagy via the ROS-EP300 pathway to eliminate intracellular M. abscessus, uncovering a potential epigenetic immune mechanism. This work provides a theoretical foundation for the clinical application of ALA-PDT in treating M. abscessus infections.